1. Field of the Invention
The present invention relates to a recombinant vector containing MJ1 gene coding an integrase derived from Enterococcus temperate bacteriophage φ FC1 and an integration method using the vector. More particularly, the present invention relates to a recombinant vector containing MJ1 gene which can make a site-specific integration in the human cell independently without other factors but not cause an excision and an integration method using the vector.
2. Description of Related Art
Enterococcus faecalis is a gram-positive anaerobic bacterium that normally occurs in the intestines of most animal from cockroaches to humans. E.faecalis KBL703 strain and its temperate phage φ FC1 was first isolated in our laboratory from a culture of the lysogenic strain of KBL703 following induction by UV irradiation (Kim et al., (1994) Mol. Cell., 4, 155-158). Bacteriophage φ FC1 has double-stranded DNA genome of approximately 40.5 Kbp, icosahedral head and a sheathless noncontractile tail. Phage φ FC1 integration into the host chromosome occurs by a site-specific mechanism. A gene that encodes a site-specific integrase, MJ1, and that is upstream of the attP site has been identified. MJ1 encodes a 465-amino-acid polypeptide with similarity in its N-terminal domain to site-specific integrases. Analysis of the DNA sequences around the attP region identified two predicted bacterium-phage junction regions (attL and attR). The corresponding bacterial attachment site (attB) was deduced from the sequences of these regions (Kim et al., (1996) Biochem. Mol. Biol, 29, 448-45412).
Integrase MJ1 mediates unidirectional site-specific integration between two DNA recognition sequences which is the phage attachment site, attP, and the bacterial attachment site, attB. To accomplish integration, temperate phage ΦFC1 encodes an integrase MJ1 that mediates integration between attP and attB. In 290 bp attB sequences, attB site has 3 bp conserved core sequences, which overlapped with 3 bp conserved core sequences of 730 bp attP. Centering around core sequences, attP and attB share their sequences in half, which become attL and attR and 3 bp core sequences is repeated (Yang et al., (2002) J. Bacteriol., 184, 1859-1864).
In gene therapy, vectors are the vehicles that are used to transfer the gene of interest to the target cells. Unfortunately, there is no such thing as a ‘good universal vector,’ all of the vectors that are currently available have both advantages and disadvantages. For example, one viral vector might be able to enter target cells very efficiently but once there invokes a strong immune response, resulting in that cell being killed by the immune system, so begin to have problem with safety in host. Accordingly, it is clear that the previously distinct boundaries between viral and non-viral vectors are becoming increasingly blurred. From these reasons, development of safe integration system into implored gene is very important. However, cruder methods prevail for placement of the introduced gene into the genome, random integration often being state of the art. Lack of control over the position of introduced DNA results in unpredictable gene expression and potentially undesirable mutagenesis of important genes. A better solution would be a method that produces efficient site-specific integration into safe locations in the target genome. In these cases, conservative site-specific integration is important in genetic engineering strategies.
Enzymes of the site-specific recombination also share high specificity, and, in addition, they act with greater efficiency. Some recombinases function with no requirement for cofactors, permitting their activity in foreign cellular environments. Recombinases such as Cre and FLP perform both integration and excision with the same target sites (Sauer, B. (1994) Curr Opin. Biotechnol., 5, 521-527). Therefore, although these recombinases efficiently perform integration in mammalian cell, the net integration frequency that they mediate is low because of the excisive back reaction. To express the desired gene stably and efficiently at specific site, problems, such as low integration efficiency and stability, must be solved.
The present inventors have identified that integrase MJ1 from φFC1 of enterococcus faecalis is a good example in the efficiency and stability.